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ADS
Basic ADS (organotypic culture) protocol from Katie and Jenn 'Preparing Dermis' *Get dermis from Shiying *Incubate in KGM or DMEM (?) for 3 days to make sure no bacteria or yeast growing *Rinse in PBS—store in PBS for up to 3 months at 4C 'Cutting Dermis' *Prepare 3x3 black square from graph paper—attach to bottom of p150 dish *Spread dermis on p150 and aspirate PBS—do not let get dry *Examine dermis—do not want too thick or too thin—med transparency with few holes *Remove patches (epidermis) from dermis—can just pick off *Identify BMZ part of the membrane (matte side) and place shiny side up (want FB on BMZ) *Cut off edges *Stretch out dermis and cut appropriate amount of squares *Place scalpel down on dermis *Use forceps to pinch between blades and pull scalpel through forceps *Transfer dermis to 12-well plate—glossy side up *Evenly spread out dermis with forceps—tightly stretched out *Use pinched forceps to stretch out dermis *Can lift plate to ensure properly spread out *Dry dermis on plate for up to 1hr—leave plate open in hood 'Seeding Fibroblasts' *Get confluent plate of FB—split 1:12—use 1ml total per piece of dermis *Add 2ml 10%FBS DMEM to each well with dermis *Add 0.5ml resuspended FB to each well with dermis *Spin for 25 min at 1000rpm *Take out 0.5ml media and add another 0.5ml FB *Rotate plate and spin again *Change media daily for 5-7 days *FB should be growing around dermis 'Lifting Dermis' *Carefully wash forceps with soap and EtOH—flame *Get ADS molds and place in p60 *Add 4.5ml 10%FBS DMEM if only lifting dermis or KGM if seeding KC *Carefully pick up top 2 corners of dermis from 12-well plate *WILL NEED TO FLIP DERMIS ONTO MOLDS *Flip dermis and place into center of ADS mold—drag from top edge into liquid and release when forceps edge on bottom of square *“Walk” dermis around edges of center square with pinched forceps *Make sure its evenly spread along square with no wrinkles *Once centered, make sure no air bubbles underneath center *Change media daily 'Seeding Keratinocytes' *Thaw matrigel in icy water to stay liquid *Get 1ml syringe and 22G needle, alcohol swab *Wipe top of matrigel bottle with alcohol swab *Flip over ADS molds so BMZ/FB side up on lid of p60 *Use needle to get 0.7ml matrigel (for 4 ADS) want 4 drops per ADS *Squirt out air bubbles *Add 4 drops of matrigel to top of ADS—make sure evenly spread *Wait ~10min for matrigel to solidify *Flip over molds and place back in 4.5ml KGM—make sure no bubbles *Split KC and count cells—want 300-500x10e3 cells *Spin down after counting and resuspend so that you add 80ul per ADS (300-500x10e3 cells in 80ul) *Pipette 160ul and add continuously ½ amount (to middle mark on pipette tip) to center of dermis *Carefully check for air bubbles beneath ADS—DO NOT MOVE TOO MUCH—want KC to settle into dermis *Change media daily 'Harvesting ADS' *Prepare cryomolds—label and add OTC to fill half *Prepare ice bucket with dry ice *Get ADS and flip over molds with forceps to remove matrigel with scalpel then return to p60 *Carefully lift dermis with forceps and place on lid of p60 *Cut off edges of ADS—do not disturb center of ADS *If sectioning in half—cut on diagonal *Carefully lift from diagonal ends and place in cryomold, long edge closest to end (labeled end of mold) *Add OTC to fill rest of cryomold—make sure ADS is flat and there are no air bubbles *Place cryomold on dry ice and wait for it to solidify *Transfer cryomolds to plastic bags and store at -80C 'Proceed with sectioning as described in cryostat protocol'